Proteins bearing a classical nuclear localization signal (cNLS) are imported into the nucleus of eukaryotic cells by the heterodimer importin a/b (also known as karyopherin a/b). This import pathway, often referred as classical, also requires the small GTPase Ran, which exists mainly bound to GTP in the nucleoplasm and to GDP in the cytoplasm. RanGTP allows movement of the import complex through the Nuclear Pore Complex, by releasing importin b from high affinity binding sites as well as releasing the import cargo into the nucleoplasm. In the past ten years a wealth of cellular, biochemical, and structural data has dramatically increased our understanding of nuclear transport. It has become evident that, in addition to the classical importin a/b- dependent nuclear import pathway, eukaryotic cells have developed a remarkable variety of alternative import pathways. Whereas we do not fully understand the need of such transport "redundancy", alternative import pathways often rely on distinct non-classical transport signals, and use diverse mechanisms of import. In this proposal we intend to investigate the molecular basis for the recognition and nuclear import of three non-classical cargos, which include the adaptor snurportin, HIV-1 Rev protein, and the transcription factor STAT1. Our research is aimed at understanding how non-classical NLSs are recognized by importin a and b and how their nuclear import is regulated, both in terms of energy requirement and kinetic of accumulation in the nucleus. Specific Aims of our work include: 1. Determining the molecular basis for the Ran and energy independent nuclear import of snurportin 2. Carry out a quantitative structural and functional analysis of HIV-1 Rev-NLS 3. Define the molecular basis for the recognition of STAT1 "dinner-specific"NLS by importin a-5.